Name: high throughput microinjections of sea urchin zygotes
Uploaded: 2014-01-21T21:00:00
Description: Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

Vi takker Santiago Suarez for kritisk læsning af manuskriptet og Betty Cowgill om støtte i fotografering. Vi takker også de anonyme anmeldere for deres kritisk feedback. Dette arbejde understøttes af University of Delaware Research Fund.

1.. Udarbejdelse af protaminsulfat (PS) Coated Dishes <ol><li> Klargør 1% opløsning af protaminsulfat (PS) ved tilsætning af 0,5 g PS i 50 ml deioniseret, destilleret vand (ddH2O) i en 50 ml konisk rør. Ryst godt ved høj hastighed på en bænk ryster ved stuetemperatur i 1-2 timer for at sikre fuldstændig opløsning af PS. Denne løsning kan opbevares ved 4 ° C i mindst 3 måneder (sørg for at opløse gel-lignende bundfald før hver brug) 3.. </li><li> Tag et ærme på 60 mm x 15 mm polys...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offerings+from+an+Urchin.">Offerings from an Urchin.</a> Dev. Biol. 358, 285-294 (2011).</li><li>McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+sea+urchins.">Evolutionary crossroads in developmental biology: sea urchins.</a> Development. 138, 2639-2648 (2011).</li><li>Sodergren, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+article+-+The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">Research article - The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Roux, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B+synthesis+is+required+for+sea+urchin+oocyte+maturation.">Cyclin B synthesis is required for sea urchin oocyte maturation.</a> Dev. Biol. 256, 258-275 (2003).</li><li>Wong, J. L., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+modifications+at+fertilization:+regulation+of+dityrosine+crosslinking+by+transamidation.">Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation.</a> Development. 136, 1835-1847 (2009).</li><li>Shuster, C. B., Burgess, D. 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Mikroinjektion er en kraftfuld teknik til at levere forskellige behandlinger såsom DNA, mRNA, rekombinante proteiner, tab af funktion og forstærkning funktions reagenser, farvestoffer og kombinationer i æg, embryoner, og celler af forskellige organismer 1-7. Dog bør flere overvejelser skal holdes i tankerne, når designe en mikroinjektion eksperiment. Det er kritisk vigtigt at overveje opløseligheden af ​​den leverede behandling og injektionsvolumen. Hvis mikroinjiceret l...

Effektiv og reproducerbar behandling levering er en af ​​de vigtigste metodiske udfordringer for forskere. Der er etableret flere metoder til forbigående levere behandling løsninger ind i de æg, embryoner og celler. Disse metoder omfatter elektroporation (baseret på en genererer forbigående porer i membranen ved hjælp af korte elektriske impulser) 1,2, lipofection (levering ved fusion af behandlings-holdige liposomer med membranen) 1, mikropartikelbombardement 1 (DNA udfældes på micron mellemstore metalpartikler, der derefter bruges til at trænge ind i cellerne ved høj hastighed) og transduktion (virus anvendes som fremføringsmiddel af transgener). I øjeblikket, mikroinjektion er den eneste metode, der besidder den fordel, at der afgives en opløsning med 100% effektivitet med minimale reagenser. Desuden kan en enkelt injektion opløsning består af en kompleks blanding af behandlinger. Denne teknik har været anvendt til vellykketly microinject æg og embryoner fra talrige arter såsom søpindsvin 3,4, zebra fisk 5, mus 6, frø 7 og kvæg 8, samt enkelte celler i vævskultur 9. Enlige blastomeres injektioner på senere udviklingsstadier er også blevet gennemført 10-12. De nuværende metoder til microinjections er baseret på tryk-injektion metode, der oprindeligt blev beskrevet af Hiramoto 10, men er blevet gjort store fremskridt i retning af en optimering af denne proces. Fremragende Mikroinjektionsteknikker er blevet beskrevet andetsteds 11, og her beskriver vi en af de konkrete metoder, der i øjeblikket anvendes til microinject søpindsvin (Strongylocentrotus purpuratus) nyligt befrugtede æg. I over et århundrede har søpindsvin været en værdifuld eksperimentel model 15,16. Søpindsvin er evolutionært tæt knyttet til chordater (inklusive os) og analyse afderes genom afslørede, at de indeholder alle de store gen familier som den menneskelige 17. De producerer et stort antal synkront udvikling af gennemsigtige embryoner, der let kan manipuleres. Brug Søpindsvin som en model organisme har søpindsvin samfund bidraget til vores forståelse af befrugtning proces 18-21, cellebiologiske processer 22-24, og de ​​regulerende netværk gen (GRNs) 25-28. Mikroinjektion i søpindsvin zygoter kræver flere trin. Først skal immobiliseres før injektioner (beskrevet nedenfor) æggene. Mikroinjektion retter overtrækkes med protaminsulfat (PS), som skaber et positivt ladet overflade, hvortil negativt ladede æg kan klæbe 3. Æggene dejellied ved inkubering i surt havvand (pH 5,15) i 10 minutter, efterfulgt af to vaske i naturligt havvand eller kunstigt havvand (pH 8,0). De dejellied æg er omhyggeligt roet i en lige linjei midten af PS-belagt skål i nærvær af 1 mM 3-aminotriazol (3-AT), som er nødvendig for at hæmme aktiviteten af ovoperoxidase der udskilles fra corticale granuler af ægget som et resultat af befrugtning 29. Dette trin er vigtigt for at forhindre hærdning af befrugtningen kuvert og lette mikroinjektion nål post. Som et alternativ til 1 mM 3-AT, kan 10 mM paraminobenzoic syre (PABA) anvendes. Injektionsvæsken er indlæst i en mikroinjektion nål ved hjælp af specialiserede microloading pipettespids og monteret på en holder fastgjort til mikromanipulator og trykenhed (figur 1). Hver nål kan anvendes til at microinject individuelle zygoter på flere forsøg på separate dage. Mikroinjektion kan udføres i 10-15 minutter, indtil zygoter hærde. De zygoter vaskes derefter med havvand og dyrket ved 15 ° C. Når embryonerne nå udrugning blastulaen tidspunkt de frigiver udrugning enzym, der fordøjer komponenter af fertilization kuvert 30 og give dem mulighed for at naturligt løsne sig fra PS-belagt fad. Hvis det er nødvendigt, kan embryonerne forsigtigt løsrevet fra skålen ved hjælp af en mund pipette eller Pasteur-pipette ved forsigtigt at blæse havvand på embryoner. Den beskrevne metode muliggør effektiv og pålidelig mikroinjektion af 100-400 nyligt befrugtede æg på et enkelt fad, hvilket giver en high-throughput metode til downstream analyser.

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

high throughput microinjections of sea urchin zygotes

Mikroinjektion i celler og embryoer er en almindelig teknik, der anvendes til at studere en bred vifte af biologiske processer. I denne fremgangsmåde en lille mængde af behandlingsopløsningen er indlæst i en mikroinjektion kanyle, der anvendes til fysisk at injicere individuelle immobiliserede celler eller embryoner. Trods behovet for indledende uddannelse til at udføre denne procedure for high-throughput levering, mikroinjektion giver maksimal effektivitet og reproducerbar afgivelse af en bred vifte af behandling løsninger (herunder komplekse blandinger af prøver) i celler, æg eller embryoner. Ansøgninger til microinjections omfatter levering af DNA-konstruktioner mRNAs, rekombinante proteiner, gevinst på funktion og tab af funktion reagenser. Fluorescerende eller kolorimetrisk farvestof tilsættes til den indsprøjtede opløsning at muliggøre øjeblikkelig visualisering af effektiv levering samt et redskab til pålidelig normalisering af mængden af ​​den leverede løsning. Den beskrevne metode giver mulighed for mikroinjektion af 100-400 Søpindsvin zygotes inden for 10-15 min.

GFP og mCherry reporterkonstruktioner blev in vitro-transkriberet og mikroinjiceret i de nyligt befrugtede æg. Embryoer blev inkuberet ved 15 ° C i 24 timer (indtil blastulaen etape) og afbildet ved hjælp af Zeiss Observer Z1 mikroskop. Injektion af reporterkonstruktioner førte ikke til nogen udviklingsmæssige defekter (figur 6). Til kvantificering af fluorescerende signaler blev billede erhvervelse udføres ved lav forstørrelse (100X) til maksimalt optage fluorescerende pixel (fi...

Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

High Throughput Microinjections of Sea Urchin Zygotes

Mikroinjektion er en almindelig teknik, der anvendes til at levere DNA-konstruktioner, mRNA&#39;er, morpholino antisense-oligonucleotider eller andre behandlinger i æg, embryoner, og celler fra forskellige arter.

High Throughput Microinjections af søpindsvin Zygoter

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

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Biology

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